Abstract

Wound healing is a complex sequence of events consisting of regeneration and repair. It has been proposed that bacteria present in wounds play a major role in delayed wound healing. The main objectives of this study are to identify the bacterial species present in wounds and to evaluate and compare the results obtained using conventional biochemical analysis and 16S rDNA sequencing. Wound swabs from 15 healing and 15 non-healing wounds were collected from the wound clinic in University Malaya Medical Centre, Malaysia. The wound microbiotas obtained by bacterial culture methods followed by biochemical tests and 16S rDNA sequencing were compared. Culture analysis and 16S rDNA sequencing both revealed that the most prevalent bacteria in wounds are Staphylococcus spp. and Pseudomons spp. The prevalence of Staphylococcus aureus was significantly higher in non-healing wounds than healing wounds (p=0.003). API® biochemical tests successfully identified all 54 isolates at species level. However, by 16S rDNA sequencing only 19 (35.2%) isolates were identified at species level and 35 (64.8%) were identified at the genus or family level. 16S rDNA sequencing however provided a better view on the phylogenetic relationships of bacterial species present in wounds.

Keywords

Bacterial identification, Biochemical tests, 16S rDNA sequencing

Introduction

Intact skin plays a major role in controlling microbiota
on the skin surface and prevents underlying tissue from
being colonised and invaded by potential pathogens.
External damage to the intact skin through wounding
provides a favourable environment for microbial colonisation
and proliferation [1]. Wound healing is a complex
process characterised by three consecutive and overlapping
stages: inflammation, proliferation and remodelling
[2]. Wound healing begins with platelet aggregation that
promotes haemostasis. This is followed by an inflammatory
cell cascade involving neutrophils, macrophages and
lymphocytes within the tissue and lastly closure of
wound via proliferation of fibroblasts and remodelling of
extracellular matrix by crosslinking of collagen and scar
maturation [3,4]. Proper circulation, nutrition, immune
state and avoidance of negative mechanical forces are
necessary for normal wound healing. Alteration in any of
these will lead to a delay in wound healing [5].

There are two types of wounds: acute and chronic. Acute
wounds are caused by external trauma such as surgical
wounds, bites, burns, minor cuts, abrasions, crushing and gun shoot injuries [1]. Acute wounds usually heal in a
very orderly and efficient manner within a predictable
time frame that leads to recovery of anatomical and functional
integrity [3,6]. Chronic wounds, however do not
heal in a predictable period and are biologically defined
by prolonged inflammation, defective re-epithelisation
and impaired matrix remodelling [7]. These wounds are
mostly caused by endogenous mechanisms associated
with predisposing conditions such as venous disease,
diabetes mellitus, central nervous system compromise,
trauma, inflammatory illnesses, metabolic abnormalities,
coagulopathies, immunosuppression, obesity, smoking
and malnutrition [8]. The presence of non-replicating
microorganisms in a wound may be considered as contamination
[9]. Contaminating microorganisms arise
from three main sources: the external environment (air or
those introduced by traumatic injury), the surrounding
skin (normal skin microfloras including Staphylococcus
epidermidis, Micrococci) and endogenous sources including
the gastrointestinal, vaginal and oral tracts [1].
Contamination might proceed to colonisation then local
infection and ultimately cause systemic disease such as
cellulitis, septicaemia or endocarditis [7,10]. Complications
of infection maybe caused directly (bacteria patho genicity) or indirectly (mediated by the immune response
in an attempt to eradicate the invading microorganisms)
[4].

In the last decade, studies of universal DNA sequences,
especially the small ribosomal subunit 16S rDNA of
bacterial genes played an important role in the accurate
identification of bacterial species and the discovery of
novel bacteria in clinical microbiology [11,12]. The 16S
sequence consists of highly conserved nucleotide sequences
which are present in all bacterial species and
variable regions that are genus- or species specific. DNA
from almost any bacterial species can be identified by
using universal PCR primers which target the conserved
regions. Bacterial species can then be revealed by comparing
the sequences of PCR products with the known
sequences in GenBank or other databases [13].

In this study, we prospectively enrolled 15 patients with
healing wounds and 15 patients with non-healing wounds
from the Wound Clinic, UMMC. The prevalence of bacterial
species in the wounds was determined. Two sets of
16S rDNA primers, PL06 and DG74 were evaluated for
their ability to identify bacteria from wound. Identity was
compared with that obtained by biochemical tests.

Materials and Methods

Sample Collection and Isolation of Bacteria

Wound swabs of 15 healing wounds and 15 non-healing
wounds were collected from subjects attending the
Wound Clinic in University Malaya Medical Centre
through a study approved by the Ethic Committee of
UMMC (Ethics Number: 962.13). Using sterile technique,
a cotton swab was rotated over the wound for 5
seconds and the tip of the swab was then broken into a
sterile transport tube containing 3ml of Phosphate Buffered
Saline. 100μl of each sample was spread on Mac-
Conkey agar and Phenylethyl Alcohol Sheep Blood
Agar. The plates were then incubated aerobically and
anaerobically at 37ºC for 4 days. Pure cultures were obtained
after several plates were streaked with single colony.

Biochemical Methods

Pure isolates were phenotypically identified through
Gram Stain and biochemical tests. API®Coryne was
used for Gram-positive bacilli, API®Strep was used for
catalase negative Gram-positive cocci, API®Staph was
used for catalase positive Gram-positive cocci, API®20E
was used for oxidase negative Gram-negative bacteria
and API®20NE was used for oxidase positive Gramnegative
bacteria. Identification was carried out according
to the manufacturer’s instructions. After 24-48 hours
incubation, the strip was read according to the reading table and the interpretation of the data was done by using
the bacterial identification software (APIwebTM).

16S rDNA Sequencing

DNA was extracted from pure bacterial colonies using igenomic
DNA extraction kit (iNTRON Biotechnology,
Korea). A single colony was picked up from the agar
plate and re-suspended into 1.5ml Phosphate Buffered
Saline. Bacteria were pelleted by centrifugation at
11337xg for 1min and suspended in 100μl of Buffer MP
and 3μl of lysozyme solution. The extraction was performed
according to the manufacturer’s instruction. Universal
primers for 16S rDNA (PL06: 5’- GGT TAA GTC
CCG CAA CGA GCG C -3’ and DG74: 5’- AGG AGG
TGA TCC AAC CGC A -3’) were used for PCR amplification.
The reaction was carried out in 25μl containing
12.5μl of 2X PCR master mix (Promega, USA), 2.5μl of
each primer (10μM), DNA template (1ng) and nuclease
free water. PCR conditions were: initial denaturation at
95ºC for 5 min, 30 cycles of denaturation at 95ºC for 1
min, annealing at 55ºC for 1 min, extension at 72ºC for 1
min and final extension at 72ºC for 10 min. Amplification
was performed on a Eppendorf Mastercycler Personal
(Eppendorf, Germany).16S rDNA sequencing was
performed on a ABI 3730XL (First Base Sdn Bhd, Malaysia).
The obtained sequences were analysed by using
Megablast®. The resulting 16S rDNA sequences of approximately
460 bases were compared with the sequences
in National Centre for Biotechnology Information
(NCBI) using the BLAST® program. Bacterial were
identified on the basis of at least 98% similarity to 16S
rDNA sequences in the database

Results

Biochemical Tests and Bacterial Identification

Two groups of patients were recruited in this study.
Group I consisted of patients with healing wounds, while
group II consisted of patients with non-healing wounds.
The demographic and clinical characteristics of the patients
are listed in Table 1. The mean age of the healers
(41.93±22.26) was significantly lower (p-value 0.0035)
than that of non-healers (63.67±14.19). All non-healing
wounds were tested positive for at least one organism by
biochemical test, while only 60% of the healing wounds
showed the presence of bacterial species. A total of 54
clinically relevant bacterial isolates were identified and
the results are presented in Figure 1. Coagulase-positive Staphylococcus aureus (56.7%) and Pseudomonas
aeruginosa (23.3%) were the most prevalent wound
bacteria. The prevalence of Staphylococcus aureus was
significantly higher in non-healing wounds than in healing
wounds (p=0.003).

Of the 54 isolates, 29 (53.7%) were identified by API®
Staph system, 10 (18.5%) were identified by API®20E
system, 9 (16.6%) were identified by API®20NE system,
5 (9.3%) were identified by API®Strep system, and 1
(1.9%) was identified by API®Coryne system. The results
obtained were scored as excellent, very good, good
and acceptable according to the manufacturer’s guide.
Primers DG74 and PL06 located at the highly conserved
16S rDNA region successfully amplified target DNA for
all 54 colonies isolated with PCR products of approximately
460 base pairs (Figure 2). While 54 isolates were
identified at species level by the API® system, only 19
(35.2%) isolates were identified by 16S rDNA sequencing to the species level, 32 (59.3%) were identified at
genus level and 3 (5.5%) were identified at family level
(Table 3). For instance, sequence comparison of an isolate
with sequences in the NCBI database showing
100.0% concordance to both Staphylococcus aureus and
Staphylococcus epidermidis. Hence this isolate can only
be identified up to its genus level, Staphylococcus spp.
Analysis of the phylogenetic tree revealed that these two
organisms are closely joined (Figure 3). For 35 isolates
identified to the family and genus level by 16S sequencing,
API® assigned the isolates to the same genus and
further identified to the species level. The identification
of 19 isolates to species level by 16S rDNA sequencing
was identical to that identified by the biochemical results

Figure 2: Phylogenetic tree constructed by MEGA5.0 based on 16S rDNA sequences amplified by PL06 and DG74 primers.
The identity of the bacteria was confirmed by biochemical tests. The scale bar represents 5% sequence divergence..

Discussion

Our findings show that the mean age of the non-healers
was significantly higher than the healers and this finding
is in agreement with the previous findings [14,15]. A
study by Harker [16] showed that systemic diseases such
as diabetes mellitus, chronic venous ulcers and arterial
ulcers which may delay wound healing are more common
in elderly. Moreover, factors such as excessive inflammatory
response, matrix degradation, impaired vascularization,
change in energy metabolism and decreased
granulation may also affect wound healing in older subjects
[15,17].

Previous studies have shown that Staphylococcus aureus
and Pseudomonas aeruginosa are the predominant microorganisms
isolated from chronic wounds [4,7,10].
This is in agreement with results from this study in which
the prevalence of Staphylococcus aureus was found significantly
higher in non-healing wounds than in healing
wounds. Athanasopoulos et al. [18] and Edwards et al.
[19] postulated that the extracellular adherence protein
(Eap) of Staphylococcus aureus played a pivotal role in
impaired wound healing by impeding the inflammatory
state and inhibiting angiogenesis in the proliferative
state. Inflammation is an important part in wound healing
and is responsible for eliminating potential pathogens
[4]. However, presence of bacterial components in
chronic wounds may stimulate excessive inflammatory
response and chronic wounds will not heal until the excessive
inflammation is reduced [3].

For decades, biochemical methods have been used as the
‘gold standard’ in identification of bacteria. To find a
effective and efficient method in identification of wound
bacteria, we compared and evaluated both biochemical
methods and 16S rDNA sequencing. Our findings revealed
that all 54 isolates were identified by the API®
systems to the species level. However, only 19 isolates
were identified to species level by 16S rDNA sequencing.
Previous studies had reported that 16S rDNA sequencing
provides genus identification in >90% of the
cases and species identification in only 65-83% cases
[12,20]. Indeed, the 16S primers (PL06 and DG74) that
were used in this study have poor discriminative power
among closely related species. For instance, Staphylococcus
aureus and Staphylococcus epidermidis showed
only <0.5% divergence (>99.5% similarity). Furthermore,
construction of a phylogenetic tree revealed little
sequence divergence between 16S rDNA sequences in
them. Identification of bacteria by 16S rDNA sequencing
depends on significant interspecies differences and small
intraspecies differences in the 16S regions [12]. Hence,
one of the limitations of 16S rDNA sequencing is inability
to identify bacterial species particularly when the
sequences have very high similarity score to the next closest match [21]. In conclusion, we confirmed that Staphylococcus aureus is the most prevalent bacteria in
non-healing wounds of older patients. 16S rDNA sequencing
provides us a better view in bacterial phylogeny
and taxonomy studies even though there are some
limitations in this method. Additional primers or probes
are necessary to target the variable region in the 16S
sequence for distinctive bacterial identification.

Acknowledgements

We gratefully acknowledge funding for the research
described in this study from University Malaya IPPP
grant (PG056-2012B). We thank the nurses of the
UMMC Wound Clinic for their knowledge and helpfulness.